Prolyl Isomerase - Evidence For Proline Isomerization

Evidence For Proline Isomerization

Methods for identifying the presence of a rate-limiting proline isomerization process in a protein folding event include:

1. Activation energies consistent with proline isomerization, which typically has an activation of about 20 kcal/mol.
2. Two-state folding kinetics indicative of both fast-folding and slow-folding populations in the unfolded or denatured state.
3. "Double-jump" assays in which proline-containing proteins are unfolded and refolded, and the population of non-native proline conformations are studied as a function of the extent of folding.
4. Acceleration of the in vitro folding rate by the addition of a prolyl isomerase.
5. Acceleration of the in vitro folding rate in mutant protein variants with one or more proline residues replaced by another amino acid.

It is important to note that not every proline peptide bond is critical to the structure or function of a protein, and not every such bond has a significant influence on folding kinetics, especially trans bonds. Furthermore, some prolyl isomerases have a degree of sequence specificity and therefore may not catalyze the isomerization of prolines in certain sequence contexts.